The identification of human T-cell lymphotropic virus-III (HTLV-III) as the probable etiologic agent of acquired immune deficiency syndrome (AIDS) has led to development of diagnostic assays for detection of antibody to HTLV-III (anti-HTLV-III) in serum or plasma. In U.S. Pat. No. 4,520,113 such an immunoassay for HTLV-III antibody is described. Subsequently, Gallo et al., Science, 224: 500-503 (1984), have reported isolation of HTLV-III virus from a majority of anti-HTLV-III positive individuals.
Previously, HTLV-III antigen has been detected in vitro by lymphocyte isolation followed by tissue culture of these lymphocytes and their co-cultivation with a human T-cell line such as HT-9 susceptible to infection with HTLV-III. Presence of HTLV-III antigen is then detected in the cell culture by testing for reverse transcriptase activity or by immunofluorescent antibody or sandwich enzyme immunoassay techniques. The problem with this method is that it is extremely technique sensitive, labor intensive and requires special laboratory facilities and two to eight week incubation periods to obtain results.
McDougal, et al., J. Immunol. Methods, 76: 171-183 (1985) have described a method for detecting lymphadenopathy-associated virus (LAV) in supernates of LAV-infected human lymphocyte cultures. LAV, like HTLV-III, is considered a prototype strain of the human retrovirus thought to be the causative agent of AIDS.
In McDougal's method, LAV is propagated in lymphoblasts, and the culture supernates are monitored for viral infection by direct immunofluorescence, enzyme-linked immunosorbent (ELISA) capture assay and by supernate reverse transcriptase activity. In the LAV-ELISA capture assay, microtiter plate reaction wells are coated with human anti-LAV IgG, the culture supernates to be tested are added to the wells, incubated and washed, and horseradish peroxidase conjugated human anti-LAV IgG is added to the wells. After incubation and washing, an o-phenylenediamine (OPD)/H.sub.2 O.sub.2 solution is added, color is developed and the optical density in the reaction walls is read in an automatic microtiter plate reader at 490 nm.
The problem with the McDougal et al. method is that it is applicable only to the assay of tissue culture supernates and, cannot be performed on human biological samples due to the likelihood of detecting false positives. The reason false positives occur in the McDougal, et al. procedure is that the human antibody used for the coating of reaction wells and in the conjugate, will react with other antigens in the human sample such as rheumatoid factor.
An easier method of detecting HTLV-III viral antigen directly from biological samples is desirable. However, it has been suggested that HTLV-III viral antigens are present in such low concentrations, if at all, in some biological samples that they would not be readily detectable. Therefore, some manipulation of whole blood samples such as lymphocyte separation followed by lysis, or small-scale lymphocyte cultivation, might be necessary in order to effectively detect viral antigen. Another potential problem in detection of HTLV-III viral antigen in biological samples is the simultaneous presence of anti-HTLV-III, leading to immune complex formation and possible masking of the HTLV-III antigens. Methods for dissociating HTLV-III antigen from immune complexes would allow for antigen detection, but would require additional processing of the sample.